Full genome sequence analysis of African swine fever virus isolates from Cameroon.

African swine fever (ASF) is a devastating disease of domestic pigs that has spread across the globe since its introduction into Georgia in 2007. The etiological agent is a large double-stranded DNA virus with a genome of 170 to 180 kb in length depending on the isolate. Much of the differences in genome length between isolates are due to variations in the copy number of five different multigene families that are encoded in repetitive regions that are towards the termini of the covalently closed ends of the genome. Molecular epidemiology of African swine fever virus (ASFV) is primarily based on Sanger sequencing of a few conserved and variable regions, but due to the stability of the dsDNA genome changes in the variable regions occur relatively slowly. Observations in Europe and Asia have shown that changes in other genetic loci can occur and that this could be useful in molecular tracking. ASFV has been circulating in Western Africa for at least forty years. It is therefore reasonable to assume that changes may have accumulated in regions of the genome other than the standard targets over the years. At present only one full genome sequence is available for an isolate from Western Africa, that of a highly virulent isolate collected from Benin during an outbreak in 1997. In Cameroon, ASFV was first reported in 1981 and outbreaks have been reported to the present day and is considered endemic. Here we report three full genome sequences from Cameroon isolates of 1982, 1994 and 2018 outbreaks and identify novel single nucleotide polymorphisms and insertion-deletions that may prove useful for molecular epidemiology studies in Western Africa and beyond.

Assessment of African swine fever vaccine candidate ASFV-G-∆MGF in a reversion to virulence study.

African swine fever (ASF) has gained panzootic dimensions and commercial vaccines are still unavailable. Recently, a series of live attenuated vaccines has raised hope for an efficacious and safe vaccine, among them "ASFV-G-∆MGF". We tested the latter in an in vivo reversion to virulence study in accordance with International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products guidelines. Upon forced animal passaging, a virus variant emerged that was associated with transient fever and an increased replication and shedding. However, all animals were healthy upon completion of the study and reversion to significant virulence was not observed. The genomic changes did not affect the recombination site but involved deletions and reorganizations in the terminal regions of the genome. Thus, our study underscores that in-depth safety characterization is needed for live ASF vaccines. For this particular candidate, additional studies should target long-term effects and transmission characteristics before thorough benefit-risk analysis can be carried out.

Characterization of a Novel African Swine Fever Virus p72 Genotype II from Nigeria.

African swine fever (ASF) is a high-consequence transboundary hemorrhagic fever of swine. It continues to spread across the globe causing socio-economic issues and threatening food security and biodiversity. In 2020, Nigeria reported a major ASF outbreak, killing close to half a million pigs. Based on the partial sequences of the genes B646L (p72) and E183L (p54), the virus responsible for the outbreak was identified as an African swine fever virus (ASFV) p72 genotype II. Here, we report further characterization of ASFV RV502, one of the isolates obtained during the outbreak. The whole genome sequence of this virus revealed a deletion of 6535 bp between the nucleotide positions 11,760-18,295 of the genome, and an apparent reverse complement duplication of the 5' end of the genome at the 3' end. Phylogenetically, ASFV RV502 clustered together with ASFV MAL/19/Karonga and ASFV Tanzania/Rukwa/2017/1 suggesting that the virus responsible for the 2020 outbreak in Nigeria has a South-eastern African origin.

Taking a Promising Vaccine Candidate Further: Efficacy of ASFV-G-ΔMGF after Intramuscular Vaccination of Domestic Pigs and Oral Vaccination of Wild Boar.

African swine fever (ASF) is a pandemic threat to the global pig industry and wild suids. A safe and efficacious vaccine could monumentally assist in disease eradication. In the past years, promising live attenuated vaccine candidates emerged in proof-of-concept experiments, among which was "ASFV-G-∆MGF". In our study, we tested the vaccine candidate in three animal experiments intramuscularly in domestic pigs and orally in wild boar. Further, a macrophage-grown vaccine virus and a virus grown on permanent cells could be employed. Irrespective of the production system of the vaccine virus, a two-dose intramuscular immunization could induce close-to-sterile immunity with full clinical protection against challenge infection. After oral immunization, 50% of the vaccinees seroconverted and all responders were completely protected against subsequent challenge. All nonresponders developed ASF upon challenge with two acute lethal infections and two mild and transient courses. The latter results show a lower efficiency after oral administration that would have to be taken into consideration when designing vaccination-based control measures. Overall, our findings confirm that "ASFV-G-∆MGF" is a most promising vaccine candidate that could find its way into well-organized and controlled immunization campaigns. Further research is needed to characterize safety aspects and define possible improvements of oral efficiency.

New Cell Lines Derived from European Tick Species.

Tick cell lines are important tools for research on ticks and the pathogens they transmit. Here, we report the establishment of ten new cell lines from European ticks of the genera Argas, Dermacentor, Hyalomma, Ixodes and Rhipicephalus originating from Germany and Spain. For each cell line, the method used to generate the primary culture, a morphological description of the cells and species confirmation by sequencing of the partial 16S rRNA gene are presented. Further molecular analysis of the two new Ixodes ricinus cell lines and three existing cell lines of the same species revealed genetic variation between cell lines derived from ticks collected in the same or nearby locations. Collectively, these new cell lines will support research into a wide range of viral, bacterial and protozoal tick-borne diseases prevalent in Europe.

African Swine Fever in Wild Boar in Europe-A Review.

The introduction of genotype II African swine fever (ASF) virus, presumably from Africa into Georgia in 2007, and its continuous spread through Europe and Asia as a panzootic disease of suids, continues to have a huge socio-economic impact. ASF is characterized by hemorrhagic fever leading to a high case/fatality ratio in pigs. In Europe, wild boar are especially affected. This review summarizes the currently available knowledge on ASF in wild boar in Europe. The current ASF panzootic is characterized by self-sustaining cycles of infection in the wild boar population. Spill-over and spill-back events occur from wild boar to domestic pigs and vice versa. The social structure of wild boar populations and the spatial behavior of the animals, a variety of ASF virus (ASFV) transmission mechanisms and persistence in the environment complicate the modeling of the disease. Control measures focus on the detection and removal of wild boar carcasses, in which ASFV can remain infectious for months. Further measures include the reduction in wild boar density and the limitation of wild boar movements through fences. Using these measures, the Czech Republic and Belgium succeeded in eliminating ASF in their territories, while the disease spread in others. So far, no vaccine is available to protect wild boar or domestic pigs reliably against ASF.

No evidence for African swine fever virus DNA in haematophagous arthropods collected at wild boar baiting sites in Estonia.

African swine fever (ASF) is a highly pathogenic viral disease affecting all Suidae, with Ornithodoros moubata complex soft ticks acting as the biological arthropod vectors of the causative agent, African swine fever virus (ASFV). While ASFV is also transmissible via direct contact, pig products and fomites, other arthropods may be involved in virus transmission and persistence. Therefore, we checked various groups of blood-feeding arthropods collected during summer 2017 in wild boar habitats on the Estonian Island of Saaremaa for the presence of ASFV. Saaremaa had the highest ASF infection prevalences in Estonia in 2017, with an incidence of 9% among hunted wild boar. In addition to ASFV, we tested for other selected pathogens. In total, 784 ticks, 6,274 culicoid biting midges, 77 tabanids and 757 mosquitoes were tested as individuals or pools. No ASFV-DNA was found in any of them although about 20% of the tick samples tested positive for swine DNA. By contrast, tick-borne encephalitis virus-RNA was detected in one out of 37 tick pools (2.7%) and Borrelia burgdorferi s.l.-DNA in 20 individual ticks and 17 tick pools (25.2% of all samples). No Schmallenberg virus was detected in the Culicoides specimens. In conclusion, we found no evidence for Ixodes ricinus ticks, Culicoides punctatus and Obsoletus complex biting midges, Aedes spp., Anopheles spp. and Culiseta annulata mosquitoes, and Haematopota pluvialis tabanids playing a role in ASFV transmission in the wild boar population in Estonia.

Joining the club: First detection of African swine fever in wild boar in Germany.

African swine fever (ASF) has spread across many countries in Europe since the introduction into Georgia in 2007. We report here on the first cases of ASF in wild boar detected in Germany close to the border with Poland. In addition to the constant risk of ASF virus (ASFV) spread through human activities, movements of infected wild boar also represent a route of introduction. Since ASF emerged in Western Poland in November 2019, surveillance efforts, in particular examination of wild boar found dead, were intensified in the regions of Germany bordering with Poland. The first case of ASF in wild boar in Germany was therefore detected by passive surveillance and confirmed on 10 September 2020. By 24 September 2020, 32 cases were recorded. Testing of samples from tissues of carcasses in different stages of decomposition yielded cycle threshold values from 18 to 36 in the OIE-recommended PCR, which were comparable between the regional and national reference laboratory. Blood swabs yielded reliable results, indicating that the method is suitable also under outbreak conditions. Phylogenetic analysis of the ASFV whole-genome sequence generated from material of the first carcass detected in Germany, revealed that it groups with ASFV genotype II including all sequences from Eastern Europe, Asia and Belgium. However, some genetic markers including a 14 bp tandem repeat duplication in the O174L gene were confirmed that have so far been detected only in sequences from Poland (including Western Poland). Epidemiological investigations that include estimated postmortem intervals of wild boar carcasses of infected animals suggest that ASFV had been introduced into Germany in the first half of July 2020 or even earlier.

A comparison of the cuticular properties of argasid and ixodid ticks: Ornithodoros moubata (Argasidae) vs. Amblyomma hebraeum and Ixodes pacificus (Ixodidae).

Ticks (Chelicerata, Ixodida) are blood-feeding ectoparasites believed to have evolved at least about 120 millions of years ago and found worldwide. However, many aspects of their unique life cycle and anatomy, including their mechanical properties, remain to be understood. Here, we compared the mechanical properties of the cuticle of the argasid tick Ornithodoros moubata to those of two species of ixodid tick, Amblyomma hebraeum and Ixodes pacificus that we explored in our earlier studies of the tick exoskeleton. Significant differences were expected given the substantial difference in life cycle, including a five-fold increase during the repeated adult blood meal for female O. moubata vs. 70- to 120-fold during the single feeding of the adult female A. hebraeum and I. pacificus. We demonstrate here that the layered structure and mechanical properties (stiffness and viscosity) of the cuticle show minor differences, but the difference in cuticle thickness is substantial. Ductility is lost during feeding; reduced pH restores ductility. Previous work suggests that this occurs in vivo in engorged ixodid ticks; there is no evidence of this occurring in vivo in O. moubata. Thinning of cuticle in O. moubata fed females is consistent with the predicted stretch of cuticle due to the blood meal; there is no evidence of cuticle synthesis during the short feeding period. Dimensional analysis suggests that the soft feel of argasid ticks is related to cuticle thickness, not cuticle stiffness. Relative to argasid ticks, the hard ixodid ticks accommodate a ca. 20-fold higher size of blood meal by starting with a thicker cuticle and growing much additional cuticle during engorgement.

African Swine Fever in a Bulgarian Backyard Farm-A Case Report.

African swine fever (ASF) is one of the most threatening diseases for the pig farming sector worldwide. As an effective vaccine is lacking, strict application of control measures is the only way to fight the disease in both industrial farms and backyard holdings. With generally low biosecurity standards, the latter are at particular risk for disease introduction and offer challenging conditions for disease control. In the following case report, we describe the overall course of an ASF outbreak in a Bulgarian backyard farm and the implemented control measures. Farm facilities and available data have been investigated to estimate the possible source, spread and time point of virus introduction. Contact with contaminated fomites entering the stable via human activities was regarded to be the most likely introduction route. The slow disease spread within the farm contributes to the hypothesis of a moderate contagiosity. As no further ASF outbreaks have been detected in domestic pig farms in the region, it could be demonstrated that successful disease control in small-scale farms can be reached. Thus, the report contributes to a better understanding of ASF in the backyard sector.

Identification and characterization of the 285L and K145R proteins of African swine fever virus.

African swine fever (ASF) is a lethal disease of domestic pigs and wild boar, against which no vaccines are available to date. The large dsDNA genome of African swine fever virus (ASFV) contains up to 167 ORFs predicted to encode proteins. The functions and antigenic properties of many of these proteins are still unknown, which impedes vaccine development. Based on the results of mass spectrometry-based proteome analyses of ASFV-infected cells, two highly abundant but previously uncharacterized viral proteins, p285L and pK145R, were investigated in detail. To this end, monospecific rabbit antisera and corresponding gene deletion mutants of ASFV were prepared. RNA and immunoblot analyses revealed that p285L is an early gene product expressed prior to viral DNA replication, whereas pK145R is a true late protein. The predicted membrane protein p285L could be localized in purified ASFV particles. In contrast, pK145R was not detectable in virions, but accumulated diffusely in the cytoplasm of infected cells. Deletion of 285L or K145R from the genome of a virulent ASFV strain from Armenia did not significantly affect spread and productive growth in a permissive wild boar lung cell line, nor in primary macrophage cultures. Future studies must elucidate, whether p285L and pK145R, although non-essential for in vitro propagation of ASFV, are relevant for replication or virulence in swine. Furthermore, it remains to be investigated whether deletion of the abundant ASFV proteins p285L or pK145R might support serological differentiation from wild-type-infected animals.

Deletion at the 5'-end of Estonian ASFV strains associated with an attenuated phenotype.

African swine fever (ASF) was introduced into the Eastern European Union in 2014 and led to considerable mortality among wild boar. In contrast, unexpected high antibody prevalence was reported in hunted wild boar in north-eastern Estonia. One of the causative virus strains was recently characterized. While it still showed rather high virulence in the majority of experimentally infected animals, one animal survived and recovered completely. Here, we report on the follow-up characterization of the isolate obtained from the survivor in the acute phase of infection. As a first step, three in vivo experiments were performed with different types of pigs: twelve minipigs (trial A), five domestic pigs (trial B), and five wild boar (trial C) were inoculated. 75% of the minipigs and all domestic pigs recovered after an acute course of disease. However, all wild boar succumbed to infection within 17 days. Representative samples were sequenced using NGS-technologies, and whole-genomes were compared to ASFV "Georgia 2007/1". The alignments indicated a deletion of 14560 base pairs at the 5' end, and genome reorganization by duplication. The characteristic deletion was confirmed in all trial samples and local field samples. In conclusion, an ASFV variant was found in Estonia that showed reduced virulence.